The focus of the laboratory is threefold: 1) delineate the genetic basis of immune deficiency, 2) study the consequence of these gene mutations on the ontogeny of hematopoetic cells, and 3) explore therapies for these disorders based on an understanding of the underlying molecular mechanism of disease. Employing a forward genetic approach, we identified mutations in the gene encoding NEMO (NF-kB essential modulator), an intracellular signaling constituent of the NF-kB pathway, results in ectodermal dysplasia with an immune defect in B cell terminal differentiation. Mutations in the zinc finger domain of NEMO block CD40 mediated activation of NF-kB and prevent B cells from undergoing class switch recombination (CSR) and APC's from synthesizing NF-kB regulated cytokines such as IL-12 or TNF-a when stimulated with CD40 ligand. Interestingly, NF-kB activation via other signaling pathways such as TNF-a or the Toll-like receptor family are intact in these patients. This is the first immunologic disorder directly attributable to NF-kB dysfunction, and is an unprecedented example of naturally occurring mutations in ligand, receptor, or intracellular signaling constituent, giving rise to a disease characterized by a defect in B cell terminal differentiation. We reason that the inherent immune deficiency in patients with hypomorphic mutations in NEMO is directly attributable to defects in signaling via NF-kB, and would be accompanied by differences in target gene expression. We have compared gene expression profiles of B cells from patients with CD40 ligand deficiency and ectodermal dysplasia with hyper IgM syndrome (XHM-ED-) as a means to identify NF-kB target genes mediated by CD40L signaling, which modulate CSR. We show XHM-ED patients are broadly defective in gene expression programs controlling proliferation, cell cycle progression, repair and ligation of DNA ends, and mRNA processing. We are now characterizing interesting genes not previously implicated in CSR and may be involved in recognizing and modifying the RNA/DNA template at the switch region. Our approach of utilizing comparative genomics technologies to the study of this rare genetic disease, has allowed us to deconstruct a complex signaling pathway essential for B cell terminal differentiation, which would otherwise be difficult to reveal. Further systematic genome wide analysis should provide new knowledge of mediators that regulate terminal B cell differentiation. To further facilitate studies of the physiologic role of NEMO signaling in development and in regulation of the immune system, we are generating knock-in mice, which harbor germline missense mutations in the NEMO gene. The primary aim of these experiments is to further elucidate the developmental and functional defects in lymphocytes and APCs. ES cells that harbor C417R NEMO mutation have been created and are awaiting blastocyst injection. The targeting constructs for another knockin mouse that harbors a different NEMO mutation, and flox NEMO mouse have also been completed and are transfection into ES cells. Over the past year, clinical protocol #00-I-006: Study of immune responses and safety of recombinant CD40 ligand trimer in X-linked hyper-IgM syndrome patients has been completed. This one center, investigator-initiated study is designed to determine if CD40L replacement therapy can reverse the core immunologic abnormalities in patients with congenital deficiency of CD40 ligand. With current therapies, the life expectancy in these patients by age twenty-five is less than 20%. We show that administration of recombinant CD40L can reconstitute deficient immune responses. Specifically, after four months of treatment on 3x/week dosing schedule (0.03 mg/kg/day), these patients' T cells demonstrate for the first time a capacity to synthesize IFN-g and TNF-a when stimulated with anti- CD3, or SEB, or SEA. Studies of cytokine production by intracellular staining demonstrate that CD40L is able to prime both the CD4 and CD8 T cell populations with in vivo administration. In addition, patients developed positive delayed type hypersensitivity reactions to candida, mumps, and KLH. Evidence of class switch recombination in B cells has been lacking, however, and patients may require higher doses or treatment for a longer period. These preliminary results are encouraging because it provides evidence that CD40L is biologically active in humans and they provide a rational basis for continuing to explore the therapeutic potential of CD40L in patients with hyper IgM syndrome.